PROJECT SUMMARY Immunotherapies targeting cancer-specific antigens (?neoantigens?) presented by Major Histocompatibility Complexes (MHC) have high potential for improving rates of long-term, disease-free survival. T cell engineer- ing technologies have found recent success in a limited number of clinical trials, and promise a new era of ef- fective treatments with greatly reduced adverse responses. Unlike melanoma, which neoantigen-targeting therapies have focused, head and neck squamous cell carcinomas (HNSCCs) have moderate-to-low tumor burdens. Thus they are less likely to present the same types of mutation-bearing antigens on their MHC mole- cules pursued by prior studies. However, new HNSCCs diagnoses are increasingly associated with human papilloma virus (HPV) infection, providing multiple types of alternative, therapeutically useful neoantigens that can be rationally targeted. A broad discovery platform compatible with multiple cancer neoantigen categories, including those induced by HPV, stands to extend T cell engineering approaches to HNSCC. The ultimate goal of this research is to define the broad range of HNSCC neoantigens a patient?s tumor presents on MHC, and the specific T cell receptors that recognize them. The main objectives of this proposal are (i) to compare HPV-dependent HNSCC antigens to other cancer-specific antigens; (ii) to test the extent to which neoantigen presentation can be induced by current HNSCC therapies; and (iii) to characterize the evolution of neoantigen- specific effector T cells and their T cell receptor repertoires in patients undergoing checkpoint blockade (PD1) therapy. In Aim 1, we will extend the approach we previously used to identify lymphoma-specific neoantigens derived from rearrangements of the immunoglobulin gene locus. By evaluating cell lines and primary HNSCC tumors with both DNA sequencing (exome and HPV-focused) and proteomic (protein expression, MHC antigen discovery) technologies, we will evaluate the extent to which HPV is a reliable source of neoantigens relative to other potential sources. De novo peptide sequencing methods we developed will allow us to discover neoanti- gens that would escape conventional proteomic search methods. In Aim 2, we will treat HNSCC cells with ra- diation, cisplatin, and interferon gamma in culture and in patient-derived mouse xenografts. We will monitor changes in neoantigen presentation induced by these treatments with the proteomic technologies used in Aim 1. In Aim 3, we will produce synthetic versions of prioritized neoantigens identified from our preliminary data and augmented by those revealed by Aims 1 and 2. We will assemble these with corresponding MHC mole- cules and in a multiplexed fashion, create panels of barcoded MHC tetramer panels. These will be used to profile T lymphocyte populations from HNSCC patients, from which clonal T cell receptors will be sequenced. Thus, our aims relieve the two major obstacles that currently limit neoantigen-targeting immunotherapies: they will provide high-throughput ways to identify HNSCC neoantigens and their cognate T cell receptors. This ap- proach should be broadly applicable to other cancers.